Borna disease virus (BDV) is an important model for the study of viral persistence in the central nervous system (CNS), a subject relevant to health because viruses can contribute to human neurologic disease. BDV is the prototype of a new family of mononegaviruses, and information obtained from studies on BDV might illuminate novel aspects of MNV biology. Our long-term goal is to elucidate the mechanisms that control BDV replication and gene expression as a prerequisite to understand the molecular bases of BDV persistence in the CNS and virally induced disturbances in brain cell functions. The experimental focus of this proposal is to functionally characterize viral and cellular proteins that contribute to the control of replication and gene expression program of BDV. Our specific aims are: 1. Detection and functional characterization of BDV predicted polypeptides p8.3, p8.4 and p165. BDV uses alternative RNA splicing and transcription termination signals to generate three mRNA species that can code for three novel predicted viral polypeptides whose expression and functions in the virus life cycle have not been studied. We will: 1) Determine whether these polypeptides are produced during BDV infection. 2) Characterize their sub-cellular and temporal expression patterns during BDV infection. 3) Examine their roles in the control of BDV RNA replication and gene expression. 2. Examine the roles of BDV p10 and Np38 polypeptides in the life cycle of BDV. We will determine: 1) The mechanisms whereby p10 inhibits viral RNA synthesis. 2) How p10 modulates its inhibitory activity in BDV-infected cells and whether p10 is required for virus propagation. 3) The mechanisms whereby Np38 contributes to optimal BDV polymerase activity, and whether Np38 is required for the generation of infectious virus and its propagation. 3. Biochemical and functional characterization of the BDV polymerase complex-host cell protein interactions. We will investigate the mechanisms and consequences of BDV polymerase complex-hCLE interaction we have identified. We will use Tandem Affinity Purification (TAP) and Mass Spectrometry approaches to identify the full array of BDV polymerase complex interacting partners. As a complementary approach we will use siRNA-based screenings to identify cellular inhibitors of the BDV polymerase complex. Candidate molecules identified by proteomic approaches and RNAi-based screens will be validated using biochemical and genetic approaches. The biological relevance of validated viruscell protein interactions will be examined in the context of the BDV MG rescue system and BDV-infected cells using a combination of biochemical, genetic and cell biology approaches.